Water-soluble bis(thiosemicarbazonato)copper(II) complexes

Article abstract of DOI:10.1071/CH10463

The synthesis of four new water-soluble bis(thiosemicarbazone) ligands and their copper(ii) complexes is presented and their potential to be new ligands for copper radiopharmaceuticals is discussed. The ligands and complexes have been characterized by a c

Full text of DOI:10.1071/CH10463

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2011, 64, 244–252
www.publish.csiro.au/journals/ajc
Water-soluble Bis(thiosemicarbazonato)copper(II)
Complexes
A
A
A
Gojko Buncic, James L. Hickey, Christine Schieber,
A
B
B
Jonathan M. White, Peter J. Crouch, Anthony R. White,
A
A
A C
,
Zhiguang Xiao, Anthony G. Wedd, and Paul S. Donnelly
A
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, Parkville, Melbourne, Vic. 3010, Australia.
Centre for Neuroscience and Department of Pathology, University of Melbourne,
Parkville, Melbourne, Vic. 3010, Australia.
B
C
Corresponding author. Email: pauld@unimelb.edu.au
The synthesis of four new water-soluble bis(thiosemicarbazone) ligands and their copper(II) complexes is presented and
their potential to be new ligands for copper radiopharmaceuticals is discussed. The ligands and complexes have been
characterized by a combination of NMR spectroscopy, mass spectrometry, and X-ray crystallography. The electro-
chemical behaviour of two of the copper(II) complexes was investigated by cyclic voltammetry and revealed that both
complexes exhibited a quasi-reversible redox process attributed to a CuII/CuI process. Two of the new ligands were
radiolabelled with positron-emitting 64Cu with a view to assessing their potential as ligands that bind radioactive copper
isotopes for application in diagnostic radiopharmaceuticals. The cellular uptake of the copper complexes was investigated
in SH-SY5Y cells.
Manuscript received: 17 December 2010.
Manuscript accepted: 17 January 2011.
Introduction
to the substituents on the diimine backbone of the ligand.[16,17]
For example, the copper complex of the ligand diacetyl-bis (N4-
methyl-3-thiosemicarbazone) (H2atsm), with two methyl sub-
stituents on the diimine backbone, CuII(atsm), is harder to
reduce than CuII(ptsm), the derivative that only possesses a
single methyl functional group on the backbone of the ligand
pyruvaldehyde-bis(N4-methyl-3-thiosemicarbazone) (H2ptsm)
(Fig. 1). Consequently, 64CuII(atsm) is only reduced in hypoxic
cells and is being investigated as a hypoxia imaging agent,[17–25]
whereas CuII(ptsm) is under investigation as a perfusion tracer,
and more recent developments have focussed on an ethyl
derivative.[7–9,26]
In general, bis(thiosemicarbazonato)copper(II) complexes
suffer from poor solubility in water or aqueous mixtures. For
example, CuII(atsm) has an octanol/water partition coefficient,
log P, of 1.48 and is insoluble in water. It is normally dissolved
in dimethyl sulfoxide for synthetic preparations.[12]
It was thought that new water-soluble bis(thiosemicarbazo-
nato)copper(II) complexes could have markedly different bio-
logical activity, and be of potential interest in the development
of copper radiopharmaceuticals with different biodistribution
when compared with more traditional derivatives such as
CuII(atsm) and CuII(ptsm). The new water-soluble ligands were
prepared using a selective transamination reaction on asym-
metric bis(thiosemicarbazone) precursors. The synthesis of
four new ligands is presented as well the synthesis of their
CuII complexes. Representative examples of the new ligands
were readily radiolabelled with positron-emitting copper-64 and
The biological activity of copper complexes of bis(thiosemi-
carbazone) ligands has led to them being investigated in models
of relevance to the treatment of cancer and Alzheimer’s
disease.[1–5] There is also interest in the use of this family of
ligands to act as delivery vehicles for radioactive isotopes in the
development of copper radiopharmaceuticals. The decay profile
of copper-64 includes positron emission (bþ; Eav 278 keV,
17.9%) and bꢀ emission (37%) with a half-life of 12.7 h, and
consequently offers the potential for both positron emission
tomography (PET) imaging and radiotherapy.[6] As a non-
invasive imaging technique, PET can provide valuable diag-
nostic information to assist in clinical diagnosis. The technique
relies on a positron-emitting tracer that is detected as it passes
through the body. The use of copper radioisotopes in radio-
pharmaceuticals is dependent on the ability to selectively
deliver the radioisotope to target tissue. One approach is to
incorporate the radioactive copper isotope into a coordination
complex. The biodistribution of the copper complex is dictated
by a variety of factors including lipophilicity, size of the com-
plex, and redox properties. A wide range of bis(thiosemicarba-
zonato) ligands have been investigated as delivery vehicles for
copper radioisotopes as they form stable (Ka ¼ 1018), neutral,
membrane permeable copper complexes.[7–15] The stable,
neutral complexes can diffuse into cells where a reducing
environment renders the complexes susceptible to intracellular
reduction (CuII to CuI). The cellular metabolism of bis(thio-
semicarbazonato)copper(II) complexes is remarkably sensitive
Ó CSIRO 2011
10.1071/CH10463
0004-9425/11/030244

RESEARCH FRONT
Water-soluble Bis(thiosemicarbazone) Ligands
245
preliminary cell uptake data in neuronal-like SH-SY5Y cells are
presented. One of the ligands presented in this paper, [H2L3]ꢀ,
and its use as a zinc sensor have been reported in a preliminary
communication.[27]
lipophilic ligands. An analogous transamination reaction was
described previously to prepare bifunctional bis(thiosemicarba-
zone) chelators.[31]
The asymmetric bis(thiosemicarbazone) central to the
selective transamination reactions (H2L1) was prepared by con-
densation of diacetyl-mono-4-methyl-3-thiosemicarbazone with
4,4-dimethyl-3-thiosemicarbazide and an acetic acid catalyst
in DMF at room temperature.[31] The reaction of H2L1 with
nucleophilic amines results in the selective displacement of
dimethylamine from the dimethyl-substituted moiety of the bis
(thiosemicarbazone). This reaction occurs under mild conditions
exclusively on the N4,N4-dimethyl thiosemicarbazone functional
group (N(CH3)2) as it contains a more electrophilic thiocarbonyl
carbon atom than the N4-monosubstituted functional group
(N–CH3). The leaving group is the secondary amine dimethyl-
amine. In this case, the nucleophilic amine is an aromatic amine
sulfonate, either sulfanilic acid to give [H2L3]ꢀ, 4-amino-3-
hydroxy-1-napthalene sulfonic acid to give [H2L5]ꢀ, or 5-
amino-2-napthalene sulfonic acid to give [H2L6]ꢀ. In each case,
the leaving amine is protonated to give the dimethylammonium
salt of the new proligands, H2NMe2[H2L3–6].
Results and Discussion
Synthesis
The stability and reduction potential of bis(thiosemicarbazonato)
copper(II) complexes is more dependent on the substituents on
the diimine backbone than modifications to the terminal N4-
substitutents. Attempts to maintain the subtle control of the CuII/
CuI redox couple of bis(thiosemicarbazonato)copper complexes
have focussed on the addition of functional groups at the terminal
N4-positions of the ligand framework.[28–30] Four new ligands
were prepared by the addition of water-solubilizing aromatic
sulfonate functional groups to the N4-position of the ligand
framework via a selective transamination reaction of asymmetric
bis(thiosemicarbazone) precursors H2atsm/m2 (diacetyl-4,4-
dimethyl-4-methyl-bis(thiosemicarbazone), H2L1) (/m2 refers to
4,40-dimethyl functional group) and H2ptsm/m2 (pyruvate-4,4-
dimethyl-4-methyl-bis(thiosemicarbazone), H2L2) with aroma-
tic amine sulfonates. The addition of aromatic sulfonate groups is
a standard approach for introducing water solubility to otherwise
The synthesis of H2L2 is presented here for the first time
(Scheme 1). A Schiff base condensation between 4-methyl-3-
ethylthiosemicarbazide and pyruvaldehyde allowed isolation of
a single product. Condensation at the ketone functional group
of pyruvaldehyde occurred selectively on the keto aldehyde
1,2-dione precursor to give 2-(4-N-methyl-3-thiosemicarba-
1
zone)pyruvaldehyde (1). The H NMR of 1 clearly identified
the regiochemistry of the condensation product, as the aldehyde
proton (d 11.18 ppm) and methyl group protons (d 1.94 ppm)
are readily identified. This product (1) may not represent the
major product of the reaction as the aldehyde functional group
would be expected to be more reactive to condensation with
Fig. 1. The structures of CuII(atsm) and CuII(ptsm).
(a)
Ϫ
ϩ
MeCN
4h, reflux 98°C
85%
(b)
H2O
1.9h, 5°C
38%
DMF
48h, rt
85%
Ϫ
ϩ
MeCN
3.5h, reflux 98°C
50%
Scheme 1. The synthesis of new sulfonated proligands (a) H2NMe2[H2L3] and (b) H2NMe2[H2L4].

RESEARCH FRONT
246
G. Buncic et al.
ϩ
Ϫ
ϩ
Ϫ
ϩ
Ϫ
ϩ
Ϫ
Fig. 2. The water soluble sulfonated CuIIL3–6 complexes prepared from asymmetric bis(thiosemicarbazone) precursors H2L1 and H2L2.
4-methyl-3-thiosemicarbazide, but 1 is readily isolated in high
purity by crystallization, albeit in relatively low yields (,30%).
Attempts to recover the product due to condensation at the
aldehyde functional group were unsuccessful. A second con-
densation reaction of 2-(4-N-methyl-3-thiosemicarbazone)-
pyruvaldehyde (1) with 4,4-dimethyl-3-thiosemicarbazide
allowed isolation of H2ptsm/m2, H2L2. This method for the
synthesis of asymmetric bis(thiosemicarbazones) derived from
pyruvaldehyde is an alternative to previously reported methods
that rely on oxidative cleavage of pyruvaldehyde dimethyl-
acetal 2-thiosemicarbazone.[9,10] The transamination reaction
of H2L2 with sulfanilic acid led to the successful preparation of
Fig. 3. An ORTEP representation (40% probability) of the molecular
Me2NH2[H2L4], which could be converted to the sodium salt
structure of Na[CuIIL3]ꢂ[(CH3)2SO]3. Solvent omitted for clarity.
by passage through a cation-exchange column.
Each ligand was characterized by NMR spectroscopy and
mass spectrometry. For each ligand, the terminal N–CH3 pro-
corresponding to [CuIILx]ꢀ with the correct isotope splitting
pattern.
tons were split into doublets (d ,3.0 ppm; 3JHH ,4.5 Hz) with
the corresponding secondary amine protons seen as quartets
The copper complex [CuIIL3]ꢀ was characterized as a
sodium salt by single-crystal X-ray crystallography (Fig. 3,
(d ,8.5 ppm). The dimethyl ammonium counterion could be
identified by a triplet (d ,2.5–2.6 ppm; 6H). Introduction of
aromatic sulfonate groups was reflected by downfield shifts of
the backbone methyl protons (from 2.1–2.2 to 2.2–2.4 ppm) and
all other signals were as expected.
Table 1). As expected, the CuII is essentially four-coordinate
distorted square planar, although a weak axial interaction with
0
˚
the sulfur atom of an adjacent molecule (Cu1–S1 2.872(1) A)
results in a tendency towards square pyramidal geometry. The
trianionic ligand coordinates through the two azamethinic
nitrogen atoms and two sulfur atoms to give a distorted square
planar geometry with a 5–5–5 chelate ring system. The Cu–S
The copper complexes of the new proligands are readily
prepared by the addition of a CuII source to the ligands (Fig. 2).
As is common with other bis(thiosemicarbazone) systems,
coordination of CuII is coupled with a double deprotonation,
but for the present cases, the ligand now becomes trianionic
to give an overall monoanionic complex. The CuII complexes
retain the brown-orange colours common to the better known
variants CuII(atsm) and CuII(ptsm).[12] For example, electronic
spectroscopy in aqueous buffer solution reveals that [CuIIL3]ꢀ
has an absorption maximum at l ¼ 460 nm (e ¼ 1.1(0) ꢁ
104 Mꢀ1 cmꢀ1), whereas [CuIIL4]ꢀ has an absorption maximum
at l ¼ 473 nm (e ¼ 1.2(0) ꢁ 104 Mꢀ1 cmꢀ1). All the CuII com-
plexes could be identified by electrospray ionization (ESI)-mass
spectrometry operating in negative-ion mode by molecular ions
˚
˚
bond lengths, Cu–S1 2.274(1) A and Cu–S2 2.263(1) A, are
similar to the analogous bond lengths in CuII(atsm), as are the
Cu–N bond distances (Table 2).[13,24] The ligand cavity appears
a little too small for CuII and the deviations from ideal square
planar geometry are evident in the S1–Cu–S1 bond angle of
109.4(4)8 and the N3–Cu–N4 bond angle of 80.3(1)8. The
deprotonation of both thiosemicarbazone limbs is reflected
˚
in the C2–S1 distance of 1.770(4) A and the C5–S2 distance
˚
of 1.757(4) A, suggesting more thiolate-like than thione-like
character.[32,33] Each complex anion associates with an adjacent
molecule through weak Cu1–S10 interactions and the sulfonate

RESEARCH FRONT
Water-soluble Bis(thiosemicarbazone) Ligands
247
Table 1. Crystallographic data
Crystal
Na[CuIIL3]ꢂ[(CH3)2SO]3
Me2NH2[CuIIL4]ꢂ(CH3)2SO
Chemical formula
Mw
C19H33CuN6O6S6Na
720.40
C16H27CuN7O4S4
573.23
Crystal system
Temperature [K]
Space group
Triclinic
130.0(2)
P1
Monoclinic
130.0(2)
P21/c
˚
a [A]
˚
b [A]
˚
c [A]
9.4755(5)
10.1109(7)
17.3555(13)
79.512(6)
79.796(5)
69.574(5)
1520.48(17)
2
7.8258(12)
8.8541(13)
35.655(5)
90
a [8]
b [8]
g [8]
93.564(3)
90
3
˚
V [A ]
2465.8(6)
4
Z
Independent reflections
Rint
5776
4337
0.0450
0.0838
R (I 4 2s(I))
wR (all data)
˚
Residual electron density (min, max) [e A
Goodness-of-fit on F2
0.0455
0.0514
0.1299
0.1119
ꢀ3
]
0.79, 0.97
1.042
ꢀ0.43, 0.62
1.038
Both CuII(ptsm) and CuII(atsm) are insoluble in water but, as
II
3
Table 2. Selected bond lengths [A] and angles [8] for Na[Cu L ]
˚
.
II
[(CH3)2SO]3 and Me2NH2[Cu L ] (CH3)2SO
4
.
intended, these new complexes with sulfonate functional groups
are very soluble in water. The solubility of the Me2NH2[CuIIL5]
salts is 45 g Lꢀ1 (approximately 10 mM) whereas the solubility
Na[CuIIL3]ꢂ[(CH3)2SO]3 Me2NH2[CuIIL4]ꢂ(CH3)2SO
of the Na[CuIIL3] salts is 410 g Lꢀ1
.
Cu–S1, Cu–S2
2.2742(11), 2.2631(10)
2.2361(11), 2.2509(11)
Cu–N3, Cu–N4 1.959(3), 1.966(3)
1.965(3), 1.957(3)
C3–C4
O2–Na
1.476(5)
2.258(3)
1.462(5)
–
Electrochemistry
Correlations between the reduction potential and likely intra-
cellular reduction of copper complexes in dimethylsulfoxide
(DMSO) have proved useful in predicting the retention of
copper inside the cell. The hypoxia selectivity of CuII(atsm) has
been explained, in part, by the more negative reduction potential
of CuII(atsm) (Em ¼ ꢀ0.60 V versus saturated calomel electrode
(SCE) SCE, where Em ¼ [Epc þ Epa]/2 and for ferrocene (Fc/
Fcþ) ¼ Em ¼ 0.54 V) when measured in anhydrous DMF at a
glassy carbon working electrode compared with Em ¼ ꢀ0.51 V
for CuII(ptsm).[12,34] Cyclic voltammetry experiments in DMSO
using tetrabutylammonium tetrafluoroborate as the supporting
electrolyte showed a quasi-reversible processes for the [CuL3]ꢀ
complex with the CuII/CuI couple (Em ¼ ꢀ0.49 V) and another
quasi-reversible process at Em ¼ 0.76 V (versus SCE). For
[CuL4]ꢀ, a quasi-reversible process attributed to the CuII/CuI
couple is at Em ¼ ꢀ0.39 V (Fig. 6). The water solubility of
[CuIIL3]ꢀ and [CuIIL4]ꢀ permitted electrochemical measure-
ments in a 100% aqueous buffer (20 mM PO24ꢀ, 100 mM NaCl).
The CuII/CuI process was less reversible in aqueous buffer when
compared with measurements in DMSO. The use of faster scan
rates (2 V sꢀ1) was required to achieve quasi-reversible CuII/CuI
couples. For [CuIIL3]ꢀ, the CuII/CuI process occurred at Em ¼
ꢀ0.51 V (versus Ag/AgCl) whereas for [CuIIL4]ꢀ, it occurred
at Em ¼ ꢀ0.42 V. Under the same conditions, potassium ferri-
cyanide had Em ¼ 0.20V.
S1–Cu–S2
S1–Cu–N3
S2–Cu–N4
N3–Cu–S2
N4–Cu–S1
N3–Cu–N4
109.04(4)
84.77(10)
83.79(10)
159.51(11)
163.63(10)
80.30(13)
109.95(4)
85.48(10)
83.79(10)
164.51(10)
166.20(10)
80.76(13)
functional group is bound to the sodium counterion (O2–Na
˚
2.257(3) A). The sodium atom is five-coordinate with two
monodentate dimethylsulfoxide molecules coordinating through
oxygen and two m2-O bridging dimethyl sulfoxide molecules.
These interactions result in an elegant infinite chain or coordina-
tion polymer (Fig. 4).
An ORTEP representation of the anion found in
Me2NH2[CuIIL4]ꢂ(CH3)2SO is shown in Fig. 5. The CuII is
four-coordinate CuN2S2 square planar. The Cu–S bonds are
slightly shorter when compared with [CuIIL3]ꢀ Cu–S1
2.251(1) A and Cu–S2 2.236(1) A, perhaps reflecting the lack
of axial interactions in [CuIIL4]ꢀ. As in the previous structure,
the deprotonation of each thiosemicarbazone limb is reflected in
˚
˚
˚
the C2–S1 and C5–S2 bond lengths of 1.763(4) and 1.768(4) A
respectively. When focussing on the ligand framework, the
most significant difference between the two structures presented
˚
is the length of the C–C backbone, C3–C4 1477(6) A in
II
3
II
4
Radiolabelling with Copper-64 and Cell Uptake Studies
˚
Na[Cu L ] and 1.462(6) A in Me2NH2[Cu L ]. The di-
methylammonium cation that originates from the leaving
group in the transamination reaction is involved in hydrogen-
bonding interactions to the sulfonate functional group.
The ligands H2L5 and H2L6 were radiolabelled with 64CuII at
room temperature in aqueous sodium acetate buffer (pH ¼ 4).
Analysis by reverse-phase HPLC with radioactivity detection

RESEARCH FRONT
248
G. Buncic et al.
O4
O6
Na
C9
O1
S3
C13
N4
C10
S6
C11
C12
C3
C4
Cu
N5
C5
O3
C8
N2
C6
C7
C1
C2
N6
N3
S2
N1 S1
Fig. 4. A representation of an infinite chain from the X-ray structure of Na[CuIIL3]ꢂ[(CH3)2SO]3 generated by axial interactions between CuII and a sulfur
atom of an adjacent complex as well sodium–sulfonate interactions.
10
H2NMe2[CuIIL4]
Na[CuIIL3]
5
0
Ϫ5
Ϫ10
Fig. 5. An ORTEP representation (40% probability) of the anion from
Me2NH2[CuIIL4]ꢂ(CH3)2SO. Solvent and counterion omitted for clarity.
Ϫ15
Ϫ0.9
Ϫ0.7
Ϫ0.5
Ϫ0.3
Potential [V]
(Fig. 7) showed the compounds were prepared with high
radiochemical purity (495%), with no uncoordinated or ‘free’
64CuII (under these conditions ‘free’ 64CuII elutes at ,2 min).
The similar retention times (9.08 and 9.32 min respectively) of
the two complexes highlights the subtle difference in lipo-
philicity attributed to the hydroxyl on H2L.[5]
Fig. 6. Cyclic voltammograms of Na[CuIIL3] (black) and H2NMe2
[CuIIL4] (grey) (1.0 mM) at a scan rate of 100 mV sꢀ1. Measurements were
performed in DMSO (10 mM Bu4NBF4) relative to Fc/Fcþ, Em ¼ 0.54 V
versus SCE.
Neutral copper complexes of bis(thiosemicarbazonato)
ligands, such as CuII(atsm) and CuII(ptsm), have been shown
to be membrane-permeable despite their poor solubility in water
or aqueous mixtures. To investigate the cell uptake of the water-
soluble bis(thiosemicarbazonato) copper(II) analogues [CuL3]ꢀ,
[CuL4]ꢀ, and [CuL6]ꢀ, SH-SY5Y cells were incubated with
the three complexes at 10-mM concentration for 1 h and intra-
cellular copper levels were measured by inductively coupled
plasma mass spectrometry (ICP-MS) (Fig. 8).
HO
N
SO3H
N
N
S
N
S
N
N
N
N
64Cu
64Cu
N
H
N
H
N
H
S
N
H
S
SO3H
3.5
3
3.5
3
2.5
2
2.5
2
As the positive control, cells were also treated with
CuII(atsm) (10 mM) and a significant increase of more than
100-fold intracellular copper levels was observed compared
with cells treated with solvent alone (DMSO). The negatively
charged complexes [CuL3]ꢀ, [CuL4]ꢀ, and [CuL6]ꢀ induced
only a two-fold increase in intracellular copper levels, when
compared with untreated cells (DMSO), similarly to the
treatment with CuSO4 (10 mM) (Fig. 8). The addition of water-
1.5
1
1.5
1
0.5
0.5
0
5
10
15
0
5
10
15
Time [min]
Time [min]
Fig. 7. (Left) Radio-HPLC of 64CuIIL5 (retention time Rt: 9.08 min) and
(right) 64CuIIL6 (Rt: 9.32 min).

RESEARCH FRONT
Water-soluble Bis(thiosemicarbazone) Ligands
249
cell-uptake properties when compared with the CuII(atsm) and
CuII(ptsm) and as such, it is likely they will show different
in vivo biodistribution and could provide an interesting variant
on the use of copper complexes of bis(thiosemicarbazones) as
radiopharmaceuticals.
1
0.9
0.8
0.7
0.6
Experimental
Crystallography
Crystals of Na[CuIIL3]ꢂ[(CH3)2SO]3 and Me2NH2[CuIIL4]ꢂ
(CH3)2SO were mounted in low-temperature oil, then flash-
cooled to 130 K using an Oxford low-temperature device.
Intensity data were collected at 130 K with an Oxford XCalibur
X-ray diffractometer with Sapphire CCD detector using Cu-Ka
0.03
0.02
0.01
0
˚
radiation (graphite crystal monochromator l ¼ 1.54184 A). Data
were reduced and corrected for absorption.[37] The structures
were solved by direct methods and difference Fourier synthesis
using the SHELX suite of programs[38] as implemented within
the WINGX[39] software. Thermal ellipsoid plots were generated
using the program ORTEP-3 integrated within the WINGX
suite of programs. CCDC reference numbers: Na[CuIIL3]ꢂ
[(CH3)2SO]3, 807829; Me2NH2[CuIIL4]ꢂ(CH3)2SO, 807828.
DMSO CuII(atsm) CuIISO4
CuIIL6
CuIIL4
CuIIL3
Fig. 8. Cell uptake studies. SH-SY5Y cells were treated with copper
complexes (10 mM) or DMSO as the control for 1 h. The copper levels were
measured in washed cell pellets by inductively coupled plasma mass
spectrometry and calculated as mg Cu per mg protein.
General
solubilizing functional groups has drastically altered the mem-
brane permeability. Similar effects were seen on a related ligand
system.[35]
All reagents and solvents were obtained from commercial
sources (Sigma–Aldrich) and used as received unless otherwise
stated. H2atsm/m2 (H2L1) and sodium diacetyl-4-methyl-40-
p-sulfonato-bis(thiosemicarbazone) (H2L3) were prepared pre-
viously. Cation-exchange chromatography was performed on
DOWEXÒ 50WX2–200 ion-exchange resin (Naþ form, 1.5ꢁ
10 cm). Elemental analyses for C, H, and N were carried out
by Chemical & MicroAnalytical Services Pty Ltd, Vic. NMR
spectra were recorded on a Varian FT-NMR 500 spectrometer
(1H NMR at 499.9 MHz and 13C{1H} NMR at 125.7 MHz) at
298 K and referenced to the internal solvent residual peak. Mass
spectra were recorded on an Agilent 6510-Q-TOF LC-MS mass
spectrometer and calibrated to internal references.
This corresponds to cellular copper levels of 0.2 and 0.1 mg
copper per mg protein for the copper complexes and CuSO4
respectively. The biodistribution of the copper complex is
dictated by a variety of factors including lipophilicity, size of
the complex, and redox properties. Lipid membranes cannot be
regarded as a homogeneous barrier but rather a heterogeneous
arrangement of amphiphilic molecules in which a structured
hydrophobic core is sandwiched between two polar surfaces.[36]
It is likely that the anionic charged copper complexes cannot
penetrate the negatively charged top surface of the membrane
bilayer. As might be anticipated, uptake into cells is facilitated
by neutral complexes.
UV-Visible Spectroscopy
UV-vis spectra were recorded on a Cary 300 Bio UV-vis
spectrophotometer, from 800 to 200 nm at 0.5-nm data intervals
with a 600-nm minꢀ1 scan rate.
Conclusions
Four new bis(thiosemicarbazone) ligands were prepared utiliz-
ing selective transamination reactions. This synthetic metho-
dology is extremely versatile and allows the preparation of
mixed bis(thiosemicarbazones) containing dissimilar thiosemi-
carbazone functional groups. The introduction of aromatic
sulfonate functional groups to bis(thiosemicarbazone) ligands
induces significant water solubility in both the ligands and their
copper complexes. The solubility of the copper complexes in
water as either their Me2NH2 or sodium salts is at least 5 g Lꢀ1
(approximately 10 mM). The copper complexes [CuIIL3]ꢀ and
[CuIIL4]ꢀ retain the quasi-reversible CuII/CuI redox couples
displayed by the ‘parent’ molecules CuII(atsm) and CuII(ptsm),
which are currently under investigation as copper radio-
pharmaceuticals. Two representative examples of this family of
ligands have been readily radiolabelled with 64CuII in high
radiochemical yield. Cell uptake studies of three of the four
copper complexes, [CuL3]ꢀ, [CuL4]ꢀ, and [CuL6]ꢀ, revealed
that the complexes were dramatically less cell-permeable than
CuII(atsm). The addition of water-solubilizing aromatic sulfo-
nate groups has completely eliminated the high membrane
permeability often associated with this family of molecules.
The presented copper complexes have markedly different
High-Performance Liquid Chromatography
Analytical RP-HPLC traces were acquired using two
chromatographic systems. System A: Agilent 1200 series HPLC
system equipped with an Agilent Zorbax Eclipse XDB-C18
column (4.6 ꢁ 150 mm) with a 1 mL minꢀ1 flow rate and UV
spectroscopic detection at 214, 220, and 270 nm. Retention
times (Rt, min) were recorded using a gradient elution method of
0–100% B over 25 min; solution A consisted of water (buffered
with 0.1% trifluoroacetic acid) and solution B consisted of
acetonitrile (buffered with 0.1% trifluoroacetic acid). System B:
Shimadzu LC-20AT HPLC system equipped with a Waters
Cosmosil C18 column (4.6 ꢁ 150 mm) with a 1-mL minꢀ1 flow
rate and a sodium iodide scintillation detector and UV-vis
detector in series (detection at 220 and 275 nm). Retention times
(Rt, min) were recorded using a gradient elution method of 0% B
for 1 min, 0–100% B over 10 min, 100–0% B over 2 min, and 0%
B for 2 min, where solution A consisted of water (buffered with
0.1% trifluoroacetic acid) and solution B consisted of aceto-
nitrile (buffered with 0.1% trifluoroacetic acid).

RESEARCH FRONT
250
G. Buncic et al.
Electrochemistry
ethanol and diethyl ether, and dried under high vacuum to give
0.69 g (85%) of a yellow solid. dH (500 MHz, [D6]DMSO) 11.21
(s, 1H, N–NH–C¼S), 10.41 (s, 1H, N–NH–C¼S), 8.49–8.44
(q, 1H, CH3–NH–C¼S), 7.77 (s, 1H, N¼C–H), 3.26 (s, 6H,
N(CH3)2), 3.01 (d, 3JHH 4.6, 3H, NH–CH3), 2.09 (s, 3H, N¼C–
CH3). 13C{1H} NMR (125.7 MHz, [D6]DMSO) 180.6 (C¼S),
178.3 (C¼S), 147.0 (C¼N), 143.4 (C¼N), 42.4 (N(CH3)2), 31.2
(NH–CH3), 11.1 (C–CH3). m/z (HRMS ESIꢀ) (Calc.) 259.0823
(259.0878) [M – H]ꢀ.
Cyclic voltammograms were recorded using an Autolab
PGSTAT100 equipped with GPES V4.9 software. Measure-
ments on the complexes were carried out at ,1 ꢁ 10ꢀ4 M in
DMSO with tetrabutylammonium tetrafluoroborate (0.1 M)
as electrolyte, using a glassy carbon disk (diameter, 3 mm)
working electrode, a Pt wire counter/electrode, and an Ag/Agþ
pseudo reference electrode (silver wire in CH3CN (NBu4BF4
(0.1 M)), AgNO3 (0.01 M)). Ferrocene was used as an internal
reference (Em(Fc/Fcþ) ¼ 0.54 V versus SCE), where Em refers
to the midpoint between a reversible reductive (Epc) and oxi-
dative (Epa) couple, given by Em ¼ (Epc þ Epa)/2.
Dimethylammonium 1-(4-N-p-Sufonato-
3-thiosemicarbazone)-2-(4-N-methyl-3-
thiosemicarbazone)pyruvaldehyde (H2NMe2[H2L4])
64Cu Radiolabelling
H2L2 (0.34 g, 1.30 mmol) and sulfanilic acid (0.20 g,
1.16 mmol) were suspended in acetonitrile (50 mL) and the
mixture was refluxed under nitrogen for 4 h. After cooling to
room temperature, a light-yellow precipitate was collected by
filtration, washed with acetonitrile and diethyl ether, and dried
under high vacuum (0.25 g, 50%). dH (500 MHz, [D6]DMSO)
12.11 (s, 1H, Ar–NH–C¼S), 10.42 (s, 1H, N–NH–C¼S), 10.02
64CuIICl2 (2.15 GBq mLꢀ1, pH 1) was purchased from ANSTO
Radiopharmaceuticals and Industrials (ARI, Lucas Heights,
NSW, Australia). The radionuclidic purity at calibration
{(64Cu)/(67Cu)} was 100% and the radiochemical purity as CuII
was 100%. The chemical purities of copper, zinc, and iron were
3.5, 0.06, and 1 mg mLꢀ1 respectively.
3
(s, 1H, N–NH–C¼S), 8.57 (q, JHH 4.5, 1H, CH3–NH–C¼S),
General Procedure
8.40–7.99 (bs, 2H, [H2N(CH3)2]þ), 7.77 (s, 1H, N¼C–H), 7.60–
7.55 (m, AA0BB0, 2H, ArH), 7.52–7.46 (m, AA0BB0, 2H, ArH),
An aliquot of 64CuCl2 (20 mL, ,43 MBq, pH 1.0) was added
to a solution containing the ligand (10 mL, 1 mg mLꢀ1 DMSO),
sodium acetate (90 mL, 0.1 M), and Milli-Q water (390 mL). The
reaction was left for 30 min at room temperature before 100 mL
of the reaction solution was injected onto a reverse-phase C18
analytical HPLC column. RP-HPLC (System B) 64CuIIL5, Rt:
9.08 min and 64CuIIL6 Rt: 9.32 min.
3
3
3.00 (d, JHH 4.6, 3H, NH–CH3), 2.55 (t, JHH 5.6, 6H,
N(CH3)2), 2.23 (s, 3H, N¼C–CH3).
Sodium 1-(4-N-p-Sufonato-3-thiosemicarbazone)-2-(4-N-
methyl-3-thiosemicarbazone)pyruvaldehyde (Na[H2L4])
Compound H2NMe2[H2L4] (0.22 g, 0.51 mmol) in water
(75 mL) was passed through a Dowex column in Naþ form
eluting with water. Fractions (5 ꢁ 30 mL) were collected and
tested for presence of ligand by mixing a few drops of the eluate
with copper(II) acetate monohydrate (,2 mg) showing intense
brown colour. Second, third, and fourth fractions were com-
bined and concentrated (,20 mL final volume) to give a highly
viscous liquid. Acetonitrile (100 mL) was added, and the result-
ing mixture was concentrated and triturated with acetonitrile
repeatedly (3 ꢁ 40 mL) to remove traces of water. A precipitate
formed that was collected by filtration washed with acetonitrile
and dried under high vacuum (80 mg, 38%). dH (500 MHz,
[D6]DMSO) 12.10 (s, 1H, Ar–NH–C¼S), 10.42 (s, 1H, N–
NH–C¼S), 10.01 (s, 1H, N–NH–C¼S), 8.57 (q, 3JHH 4.5, 1H,
CH3–NH–C¼S), 7.77 (s, 1H, N¼C–H), 7.60–7.55 (m, AA0BB0,
2H, ArH), 7.50–7.46 (m, AA0BB0, 2H, ArH), 3.00 (d, 3JHH 4.6,
3H, NH–CH3), 2.23 (s, 3H, N¼C–CH3). 13C{1H} NMR
(125.7 MHz, [D6]DMSO) 178.1 (C¼S), 176.1 (C¼S), 147.0
(C¼N), 145.5 (ArC), 143.4 (C¼N), 138.8 (ArC), 125.4 (ArCH),
124.8 (ArCH), 31.0 (NH–CH3), 11.2 (C–CH3). m/z (HRMS
ESIꢀ) (Calc.) 387.0405 (387.0368) [M – H]ꢀ.
Synthetic Procedures
2-(4-N-Methyl-3-thiosemicarbazone)pyruvaldehyde
To a stirred solution of pyruvaldehyde (20mL, 40% w/w,
133 mmol) in water (30 mL) acidified with hydrochloric acid
(12 M, two drops) and cooled to 08C was added 4-methyl-3-
thiosemicarbazide (2.65 g, 25.2 mmol) portionwise over 75 min.
After maintaining the same temperature for a further 40 min,
chloroform (150 mL) and water (100mL) were added to the
reaction and the organic phase was separated. Subsequent extracts
of the aqueous phase (chloroform, 2 ꢁ 50 mL) were combined
with the organic phase, dried over magnesium sulfate and con-
centrated under vacuum. Pentane was added to a point of turbidity
and the resulting mixture was stored in the freezer for 1 h. The
precipitate that formed was collected by filtration, washed with
pentane and air-dried on the filter to give 1.10 g (27%) of a light
yellow solid. A second crop (0.48 g, 11%) was also recovered. dH
(500 MHz, [D6]DMSO) 11.18 (s, 1H, O¼CH), 9.36 (s, 1H, N–
3
NH–C¼S), 9.05–8.95 (bm, 1H, CH3–NH–C¼S), 3.03 (d, JHH
4.6, 3H, NH–CH3), 1.94 (s, 3H, N¼C–CH3). 13C{1H} NMR
(125.7 MHz, [D6]DMSO) 191.6 (C¼O), 179.0 (C¼S), 145.4
(C¼N), 31.2 (NH–CH3), 9.1 (C–CH3). m/z (high resolution
(HR)MS ESIꢀ) (Calc.) 158.0394 (158.0466) [M – H]ꢀ.
Dimethylammonium Diacetyl-4-(4-amino-
3-hydroxynaptholene-p-sufonato)-40-methyl-bis
(3-thiosemicarbazone) (H2NMe2[H2L5])
H2L1 (0.10 g, 0.36 mmol) and 4-amino-3-hydroxy-1-naptha-
lene sulfonic acid (0.08 g, 0.36 mmol) were suspended in aceto-
nitrile (20 mL) and the mixture was refluxed under nitrogen
overnight. After cooling to room temperature, a colourless
precipitate was collected by filtration, washed with acetonitrile
and diethyl ether, and dried under high vacuum (0.14 g, 82%).
dH (500 MHz, [D6]DMSO) 10.59 (s, 1H), 10.25 (s, 1H), 9.73–
9.69 (m, 2H), 8.77 (d, 3JHH 8.6, 1H, ArH), 8.46 (d, 3JHH 4.5, 1H,
CH3–NH–C¼S), 8.26–8.18 (br s, 2H, [H2N(CH3)2]þ), 7.81 (s,
1-(4,4-N-Dimethyl-3-thiosemicarbazone)-2-(4-N-
methyl-3-thiosemicarbazone)pyruvaldehyde (H2L2)
To
2-(4-N-methyl-3-thiosemicarbazone)pyruvaldehyde
(0.50 g, 3.10 mmol) and 4,4-dimethyl-3-thiosemicarbazide
(0.38 g, 3.20 mmol) in dimethylformamide (30 mL) was added
acetic acid (glacial, three drops). The mixture was stirred at
room temperature under nitrogen for 48 h followed by addition
of water (50 mL) and chilling on ice for 15 min. A yellow
precipitate was collected by filtration, washed with water,

RESEARCH FRONT
Water-soluble Bis(thiosemicarbazone) Ligands
251
1H, ArH), 7.63 (d, 3JHH 8.4, 1H, ArH), 7.42 (dd, 3JHH 8.1, 7.0,
1H, ArH), 7.33–7.30 (m, 1H, ArH), 3.09 (d, 3JHH 4.5, 3H, NH–
CH3), 2.58 (t, 3JHH 5.5, 6H, N(CH3)2), 2.34 (s, 3H, N¼C–CH3).
13C{1H} NMR (125.7 MHz, [D6]DMSO) 180.0 (C¼S), 179.4
(C¼S), 150.5, 149.3, 149.1, 145.1, 133.7, 128.5, 126.5, 124.9,
123.1, 120.1, 119.0, 118.5, 35.3 (N(CH3)2), 32.1 (NH–CH3),
12.8 (C–CH3), 12.6 (C–CH3). m/z (HRMS ESIꢀ) (Calc.)
467.0635 (466.92581) [M – H]ꢀ.
monohydrate (20 mg, 0.10 mmol) in dimethylformamide
(1 mL) and the resulting mixture was left to stir under nitrogen
at room temperature for 4 h. Excess diethyl ether was added and
the resulting dark-red precipitate was collected by filtration,
washed with diethyl ether, and dried in air (30 mg, 52%).
m/z (HRMS ESIꢀ) (Calc.) 527.8610 (527.9769) [M – H]ꢀ.
Rt 9.24 min.
Dimethylammonium Diacetyl-4-(5-amino-
2-napthalenesufonato)-40-methyl-bis(3-
Dimethylammonium Diacetyl-4-(5-amino-
2-napthalenesufonato)-40-methyl-bis(3-
thiosemicarbazone) (H2NMe2[H2L6])
thiosemicarbazonato)copper(II) (H2NMe2[CuIIL6])
To H2NMe2[H2L6] (54 mg, 0.11 mmol) dissolved in
dimethylformamide (2 mL) was added copper(II) acetate mono-
hydrate (22 mg, 0.11 mmol) in dimethylformamide (1 mL) and
the resulting mixture was left to stir under nitrogen at room
temperature for 12 h. Excess diethyl ether was added and the
resulting dark-red precipitate was collected by filtration washed
with diethyl ether and dried in air (45 mg, 74%). m/z (HRMS
ESIꢀ) (Calc.) 511.9725 (511.9820) [M – H]ꢀ. Rt 9.60 min.
H2L1 (0.2 g, 0.7 mmol) and 5-amino-2-naphtalene sulfonic
acid (0.16 g, 0.7 mmol) were suspended in acetonitrile (25 mL)
and the mixture was refluxed under nitrogen for overnight. After
cooling to room temperature, the precipitate was collected by
filtration, washed with acetonitrile and diethyl ether, and dried
under high vacuum (0.2 g, 61%). dH (500 MHz, [D6]DMSO)
10.68 (s, 1H, NH–C¼S), 10.27 (br s, 2H, NH–C¼S), 8.44–8.42
(m, 1H, CH3–NH–C¼S), 8.25–8.12 (m, 3H, ArH, [H2N
(CH3)2]þ), 8.00–7.95 (m, 1H), 7.80–7.72 (m, 2H, ArH), 7.58–
Cell Culture and Inductively Coupled Plasma
Mass Spectrometry
3
7.51 (m, 2H, ArH), 3.05 (d, JHH 4.6, 3H, NH–CH3), 2.55 (t,
3JHH 5.6, 6H, N(CH3)2), 2.31 (s, 3H, N¼C–CH3). 13C{1H}
NMR (125.7 MHz, [D6]DMSO) 179.6 (C¼S), 179.4 (C¼S),
150.1, 149.0, 146.5, 136.5, 133.8, 131.0, 128.5, 127.5, 126.7,
125.2, 125.0, 123.8, 35.3 (N(CH3)2), 32.2 (NH–CH3), 12.9
(C–CH3), 12.6 (C–CH3). m/z (HRMS ESIꢀ) (Calc.) 451.0686
(450.9350) [M – H]ꢀ.
SH-SY5Y cells were grown in DMEM:F12 media supple-
mented with 10% (v/v) FBS, 50 mM Hepes, non-essential
amino acids (Invitrogen), and penicillin–streptomycin sulfate
(Invitrogen). Cultures were maintained at 378C in a humidified
incubator with 5% (v/v) CO2 and passaged every 5–6 days
at a dilution of 1:10. For cell uptake studies, cells were seeded
into 6-cm plates. Once the cells had reached 80% confluency,
the media were removed by aspiration and replaced with
fresh, FBS-free media supplemented with 10 mM copper
complexes. The compounds were prepared freshly as 10 mM
stock solutions in DMSO, and control treatments therefore
received an equivalent volume of DMSO. After treating for 1 h,
cells were harvested by scraping into the treatment media
and pelleting by centrifugation (1000g, 5 min). Pelleted cells
were rinsed three times with PBS. A small aliquot from each
sample was used to determine total cellular protein, and the
remaining sample analysed for cellular Cu using ICP-MS as
described previously.[40] Cellular metal levels were normalized
for total cellular protein and are expressed as mg Cu per mg
protein.
Sodium Diacetyl-4-p-sulfonato-40-methyl-bis(3-
thiosemicarbazonato)copper(II) (Na[CuIIL3])
To Na[H2L3] (46 mg, 0.11 mmol) in dimethylformamide
(2mL) was added copper(II) acetate monohydrate (21 mg,
0.12 mmol) in dimethylformamide (1 mL) and the resulting mix-
ture was left to stir under nitrogen for 12 h at room temperature.
Diethyl ether (20mL) was added, resulting in precipitation of a
dark-red solid, which was collected by filtration, washed with
diethyl ether, and dried under high vacuum (34 mg, 64%). Anal.
Calc. for [C13H15N6O3S3Cu]Na: C 32.13, H 3.11, N 17.29, Na
4.73. Found C 31.79, H 3.71, N 16.65, Na 5.11%. m/z (HRMS
ESIꢀ) (Calc.) 461.9668 (461.9664) [M – H]ꢀ. Rt 8.26 min.
Dimethylammonium Pyruval-1-(4-N-p-sufonato)-2-
(4-N-methyl)-bis(3-thiosemicarbazonato)copper(II)
(H2NMe2[CuIIL4])
Acknowledgements
The Australian Research Council and National Health and Medical Research
Council are acknowledged for financial support. We thank Irene Volitakis
for her expertise in ICP-MS of cell lysates and Brett Paterson for assistance
with radiolabelling.
To H2NMe2[H2L4] (0.10 g, 0.23 mmol) dissolved in
dimethylformamide (2 mL) was added copper(II) acetate mono-
hydrate (50 mg, 0.23 mmol) in dimethylformamide (1 mL) and
the resulting mixture was left to stir under nitrogen at room
temperature for 4 h. Excess diethyl ether was added and the
resulting dark-red precipitate was collected by filtration, washed
with diethyl ether and dried in air (0.11 g, 97%). Anal. Calc. for
[C14H21N7O3S3Cu]H2NMe2ꢂDMF: C 35.75 (35.94), H 4.93
(4.97), N 19.54 (19.72). m/z (HRMS ESIꢀ) (Calc.) 447.9551
(447.9507) [M – H]ꢀ. Rt 8.19 min.
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